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Diane Nava, Tatiana Lupashina, Bhavna Antony, Li Zhang, Michael David Abramoff, Christine Frances Wildsoet; Immunolesioning of glucagonergic amacrine cells in the chick retina . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2179.
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© ARVO (1962-2015); The Authors (2016-present)
Neurotoxins have been used in myopia research to ablate inner retinal cells in order to study their contributions to eye growth and refractive error regulation, although those used in past studies have been relatively unselective. The purpose of this study is to investigate immunolesioning as a potential tool to selectively ablate glucagon amacrine cells (GACs) in the chick and to compare its selectivity to previous methods.
A Saporin immunotoxin conjugated to a primary anti-glucagon antibody was injected intravitreally in the left eyes of 7-day old chicks as a 10uL solution in one of 5 concentrations (0.125, 0.25, 0.5, 0.75 or 1 uM).<br /> <br /> TUNEL staining (Roche) was used to determine the distribution of apoptotic cells and immunohistochemistry on vertical sections used to assess changes in the glucagonergic cell population.<br /> <br /> Optical coherence tomography imaging was used to investigate changes in the retina and choroid, and flash electroretinograms (ERGs) were recorded to assess changes in retinal function 4, 6 and 9 days after injection.
The maximum loss of GACs was seen with the 1 uM concentration and for concentrations lower than 1 uM, the central retina seems to be more affected than peripheral retina, where GAC immunoreactivity in the IPL/INL boundary was more apparent than in the center.<br /> <br /> The 1uM concentration significantly attenuated the photopic negative response of the flash ERG at both 4 and 7 days (p=0.0236 & p=0.0393), with no significant effect on b-wave and a-wave amplitudes. The peak of the flash ERG at approximately 200 ms was also significantly attenuated at 4 days after injection (p=0.03), but not at later time points.<br /> <br /> With the 1 uM concentration, total retinal thickness was not significantly reduced in injected eyes at any time point, while choroidal thickness underlying the area centralis was significantly increased compared to values for the contralateral eyes at both 6 and 9 days post injection (p=0.0475 & p=0.04097 respectively).
The above immunotoxin conjugate, injected intravitreally, appears to more selectively lesion GACs in the chick retina than previously tested neurotoxins, as evidenced by histological as well as retinal structural and functional data, and thus represents a suitable tool for investigating the role of GACs in eye growth regulation. The finding of choroidal thickening in lesioned eyes is a novel finding that warrants further investigation.
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